Ex vivo and in vivo expression of the thrombomodulin gene for the treatment of cardiovascular and peripheral vascular diseases

ABSTRACT

The present invention relates to methods and compositions for treatment of cardiovascular and peripheral vascular diseases using ex vivo and in vivo gene delivery technologies. One aspect of the present invention relates to a method for treating a vascular disease by introducing a DNA sequence encoding a TM protein or its variant into a segment of a blood vessel ex vitro using a gutless adenovirus vector. Another aspect of the present invention is to provide a method to deliver a gutless adenovirus vector carrying a DNA sequence encoding a TM protein or its variant using a stent.

[0001] This application claims priority from U.S. ProvisionalApplication Serial No. 60/430,099 filed Dec. 2, 2002. The entirely ofthat provisional application is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention is directed to methods and compositions forthe treatment of cardiovascular and peripheral vascular diseases, and inparticular, is directed to methods and compositions for ex vivo and invivo expression of the thrombomodulin gene using gutless adenovirusvector.

BACKGROUND OF THE INVENTION

[0003] Atherosclerosis is one of the chief causes of morbidity andmortality in the United States and many other countries of the world.(Zuckerbraun et al., Arch Surg. 137:854-861 [2002]; Kibbe et al., CircRes. 86:829-33 [2000]). This process can result in limiting the flow ofblood to the heart, kidneys and the peripheral vessels, to name a few.Current approaches to the treatment of lesions in the arteries includecoronary artery bypass graft (CABG) surgery and angioplasty with orwithout the placement of a stent. The latter may serve as a vehicle fordrug delivery, as is currently being tested in clinical trials. A numberof pharmacological agents that affect platelet function or provideanticoagulant properties have so far failed to reduce re-occlusion orintimal hyperplasia. (Kibbe et al., Circ Res. 86:829-33 [2000]).

[0004] Cardiovascular diseases, however, are the result of complexpathophysiologic processes that involve the expression of many proteinsand molecules that can adversely affect the grafted vessel (Shears etal., J Am Coll Surg., 187(3):295-306 [1998]; Ross et al., Nature,362:801-9 [1993]). Approximately 15-30% of patients receiving veingrafts for coronary or peripheral vascular disease require follow-uptreatment, either in the form of angioplasty or new grafts.

[0005] Thrombomodulin (TM) is an integral membrane glycoproteinexpressed on the surface of endothelial cells (Sadler et al., TrhombHaemost., 78:392-95 [1997]). It is a high affinity thrombin receptorthat converts thrombin into a protein C activator. Activated protein Cthen functions as an anticoagulant by inactivating two regulatoryproteins of the clotting system, namely factors Va and VI[I]a (Esmon etal., Faseb J., 9:946-55 [1995]). The latter two proteins are essentialfor the function of two of the coagulation proteases, namely factors Ixaand Xa. TM thus plays an active role in blood clot formation in vivo andcan function as a direct or indirect anticoagulant.

[0006] There are several other proteins or enzymes that have shown toreduce the process of intimal hyperplasia, whose evolution is the causeof late graft failure. For instance, Nitric oxide synthase, an enzymeexpressed by endothelial cells has been shown in animal models toinhibit intimal hyperplasia, especially the inducible enzyme (iNOS)(Salmaa et al., Lancet, 353:1729-34 [1999]; Palmer et al., Nature,327:524-26 [1987]; Kubes et al., PNAS USA., 88:4651-5 [1991]).

[0007] Animal studies shown that cytoxic gene transfection utilizing theHerpes Simplex Virus thymydine kinase gene delivered via an adenoviralvector was able to inhibit intimal hyperplasia (Steg et al.,Circulation, 96:408-11 [1997]).

[0008] Vascular endothelial growth factor (VEGF), basic fibroblastgrowth factor (bFGF) and platelet derived growth factor (PDGF) have allbeen shown to promote reendothelization and enhance the healing ofvascular injury and help limit intimal hyperplasia. (Ban Bellle et al.,Biochem Biophs Res Commun., 235:311-16 [1997]; Salyapongse et al.,Tissue Engineering 26(4):663-76 [1999]).

[0009] A gene therapy approach is currently under clinicalinvestigation. It involves the injection, directly into heart muscles,of an adenoviral vector delivery system containing the gene for theexpression of vascular endothelial growth factor (VEGF). This is beingtested in patients whose coronary vessels are not amenable to standardgrafting procedures. However, some recent adverse clinical eventsdemonstrated that injection of large quantities of adenovirus vectors isassociated with significant risks. Accordingly, there still exist a needfor a method to effectively introduce therapeutic genes, such as TM,into vascular tissues.

SUMMARY OF THE INVENTION

[0010] The present invention relates to a method and composition fortreating vascular diseases using gene delivery technologies. One aspectof the present invention relates to a method for treating a vasculardisease in a mammal comprising infecting a segment of a blood vessel invitro using a gutless adenoviral vector which comprises a polynucleotideencoding a thrombomodulin protein or its variant; and grafting thevirus-treated blood vessel in said mammal, wherein said thrombomodulinprotein or its variant is expressed in a amount sufficient to reducere-occlusion or intimal hyperplasia in the grafted blood vessel.

[0011] Another aspect of the invention relates to a method for treatinga vascular disease by evacuating a clot in a blood vessel, isolating asegment of blood vessel around the evacuating site with a ballooncatheter and infecting the segment of blood vessel in vivo using agutless adenoviral vector comprising a polynucleotide encoding athrombomodulin protein or its variant; wherein the thrombomodulinprotein or its variant is expressed in a amount sufficient to reducere-occlusion or intimal hyperplasia in the infected segment of the bloodvessel.

[0012] Another aspect of the present invention pertains to a method toadminister a therapeutically effective amount of a gutless adenovirusvector into a segment of a blood vessel in vivo using a stent, whereinsaid gutless adenovirus vector is capable of expressing a thrombomodulinprotein or a variant of the thrombomodulin protein.

[0013] Yet another aspect of the present invention pertains to apharmaceutical composition containing a gutless adenovirus capable ofexpressing thrombomodulin protein or a variant of the thrombomodulinprotein and a pharmaceutically acceptable carrier, the gutlessadenovirus is produced using a shuttle vector comprising a pBR322replication origin, a selectable marker gene, an adenovirus leftinverted terminal repeat, an adenovirus encapsidation signal, anintronic sequence, and an adenovirus right inverted terminal repeat.

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIG. 1 is a schematic drawing of an embodiment of the backboneshuttle vector of the present invention.

[0015]FIG. 2 is the DNA sequence (SEQ ID NO:1) of the gutless backboneshuttle vector.

[0016]FIG. 3 is the full length amino acid sequence (SEQ ID NO:2) ofhuman thrombomodulin.

[0017]FIG. 4 is the full length DNA sequence (SEQ ID NO:3) encodinghuman thrombomodulin.

[0018]FIG. 5 is the DNA sequence (SEQ ID NO:4) of the expressioncassette encoding human thrombomodulin.

[0019]FIG. 6 is the DNA sequence (SEQ ID NO:5) of the CMV promoter ofthe expression cassette encoding the human thrombomodulin.

[0020]FIG. 7 is the cDNA (SEQ ID NO:6) of the human thrombomodulin gene.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The practice of the present invention will employ, unless otherwise indicated, conventional methods of histology, virology,microbiology, immunology, and molecular biology within the skill of theart. Such techniques are explained fully in the literature. Allpublications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

[0022] The primary object of the present invention is to provide methodsfor treating vascular diseases using gene delivery technologies. Oneaspect of the present invention relates to a method for treating avascular disease by introducing a DNA sequence encoding a TM protein orits variant into a segment of a blood vessel in vitro using a gutlessadenovirus vector and grafting the virus-treated vessel in a patientaffected by a vascular disease. The virus-mediated TM expression reducesre-occlusion and intimal hyperplasia in the grafted vessel. This ex vivoapproach eliminates the need to inject a large quantity of virus into apatient and hence significantly reduces the viral-related toxicity.

[0023] In one embodiment, the method is used for a coronary arterybypass. In another embodiment, the method is used for the treatment ofperipheral vascular diseases. In yet another embodiment, the method isused for the for the maintenance of vein access in renal dialysispatients.

[0024] Another object of the present invention is to provide a method todeliver a gutless adenovirus vector carrying a DNA sequence encoding aTM protein or its variant using a stent. The viral vector is embedded inthe stent and is released only at a treatment site. Since the viralinfection is restricted at the treatment site and the surrounding area,only a small amount of the virus is needed and the virus-relatedtoxicity is reduced.

[0025] Yet another object of the present invention pertains to a gutlessadenovirus carrying a TM gene. In one embodiment, the gutlessadenovirus, which contains a regulatory element operably linked to a DNAsequence encoding a TM protein or its variant and a polyA sequence, isproduced using a novel shuttle vector containing a pBR322 replicationorigin, a selection marker, an adenovirus left inverted terminal repeat,an adenovirus encapsidation signal, a stuffer sequence, and anadenovirus left inverted terminal repeat.

[0026] In one embodiment, the regulatory element is a constitutivepromoter such a CMV promoter and RSV promoter. In another embodiment,the regulatory element is an inducible promoter.

[0027] The forth object of the present invention is to provide apharmaceutical composition which comprises an effective amount ofgutless adenovirus carrying a TM gene of the present invention and apharmaceutically acceptable carrier. Such compositions may be liquids orlyophilized or otherwise dried formulations and may further includediluents of various buffer content, (e.g., Tris-HCl, acetate, phosphate)pH and ionic strength, additives such as albumin and gelatin to preventabsorption to surfaces, detergents (e.g., Tween 20, Tween 80, PluronicF68, bile acid salts), solubilizing agents (e.g., glycerol, polyethyleneglycerol); anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),and preservatives (e.g. Thimerosal, benzyl alcohol, parabens).

[0028] In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

[0029] “Gene transfer” or “gene delivery” refers to methods or systemsfor reliably introducing a particular nucleotide sequence (e.g., DNA)into targeted cells. The introduced nucleotide sequences may persist invivo in episomal forms or integrate into the genome of the target cells.Gene transfer provides a unique approach for the treatment of acquiredand inherited diseases, and a number of systems have been developed inthe art for gene transfer into mammalian cells. See, e.g., U.S. Pat. No.5,399,346.

[0030] As used herein, the term “effective amount” refers to a level ofinfection which brings about at least partially a desired therapeutic orprophylactic effect in an organ or tissue infected by the method of thepresent invention. The infection with an effective amount of the vectorcarrying genetic material of interest can then result in themodification of the cellular activities, e.g., a change in phenotype, inan organ or a tissue that has been infected by the method of the presentinvention. In a preferred embodiment, the infection with an effectiveamount of the vector carrying genetic material of interest results inmodulation of cellular activity in a significant number of cells of aninfected organ or a tissue.

[0031] A gene transfer “vector” refers to any agent, such as a plasmid,phage, transposon, cosmid, chromosome, liposome, DNA-viral conjugates,RNA/DNA oligonucleotides, virus, bacteria, etc., which is capable oftransferring gene sequences into cells. Thus, the term includes cloningand expression vehicles including “naked” expression vectors, as well asviral and non-viral vectors. A vector may be targeted to specific cellsby linking a target molecule to the vector. A targeting molecule is anyagent that is specific for a cell or tissue type of interest, includingfor example, a ligand, antibody, sugar, receptor, or other bindingmolecule. The invention is also intended to include such other forms ofvectors which serve equivalent functions and which become known in theart subsequently hereto.

[0032] The term “expression control element” or “regulatory element”refers collectively to promoter sequences, polyadenylation signals,transcription termination sequences, upstream regulatory domains,origins of replication, internal ribosome entry sites (“IRES”),enhancers, and the like, which collectively provide for the replication,transcription and translation of a coding sequence in a recipient cell.Not all of these control sequences need always be present so long as theselected coding sequence is capable of being replicated, transcribed andtranslated in an appropriate host cell.

[0033] The term “promoter” is used herein in its ordinary sense to referto a, DNA regulatory sequence that are sufficient for RNA polymeraserecognition, binding and transcription initiation. Additionally, apromoter includes sequences that modulate the recognition, binding andtranscription initiation activity of RNA polymerase. Such sequences maybe cis acting or may be responsive to trans acting factors. Dependingupon the nature of the regulation, promoters may be constitutive orregulated. Examples of promoters are SP6, T4, T7, SV40 early promoter,cytomegalovirus (CMV) promoter, mouse mammary tumor virus (MMTV)steroid-inducible promoter, Moloney murine leukemia virus (MMLV)promoter, phosphoglycerate kinase (PGK) promoter, muscle creatine kinase(MCK) promoter, myosin promoter, (α-actin promoter and the like.

[0034] The term “transduction” denotes the delivery of a DNA molecule toa recipient cell either in vivo or in vitro, via a replication-defectiveviral vector, such as via a recombinant adenovirus.

[0035] “Operably linked” refers to an arrangement of elements whereinthe components so described are configured so as to perform their usualfunction. Thus, control elements operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol elements need not be contiguous with the coding sequence, solong as the function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

[0036] The term “primer” refers to an oligonucleotide which is capableof acting as a point of initiation of synthesis when placed underconditions in which primer extension is initiated. An oligonucleotide“primer” may occur naturally, as in a purified restriction digest or maybe produced synthetically.

[0037] A primer is selected to be “substantially” complementary to astrand of specific sequence of the template. A primer must besufficiently complementary to hybridize with a template strand forprimer elongation to occur. A primer sequence need not reflect the exactsequence of the template. For example, a non-complementary nucleotidefragment may be attached to the 5′ end of the primer, with the remainderof the primer sequence being substantially complementary to the strand.Non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence has sufficient complementaritywith the sequence of the template to hybridize and thereby form atemplate primer complex for synthesis of the extension product of theprimer.

[0038] “Hybridization” methods involve the annealing of a complementarysequence to the target nucleic acid (the sequence to be detected). Theability of two polymers of nucleic acid containing complementarysequences to find each other and anneal through base pairing interactionis a well-recognized phenomenon. The initial observations of the“hybridization” process by Marmur and Lane, PNAS USA 46:453 (1960) andDoty et al., PNAS USA 46:461 (1960) have been followed by the refinementof this process into an essential tool of modern biology.

[0039] The complement of a nucleic acid sequence as used herein refersto an oligonucleotide which, when aligned with the nucleic acid sequencesuch that the 5′ end of one sequence is paired with the 3′ end of theother, is in “antiparallel association.” Certain bases not commonlyfound in natural nucleic acids may be included in the nucleic acids ofthe present invention and include, for example, inosine and7-deazaguanine. Complementarity need not be perfect; stable duplexes maycontain mismatched base pairs or unmatched bases. Those skilled in theart of nucleic acid technology can determine duplex stabilityempirically considering a number of variables including, for example,the length of the oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

[0040] Stability of a nucleic acid duplex is measured by the meltingtemperature, or “Tm.” The Tm. of a particular nucleic acid duplex underspecified conditions is the temperature at which on average half of thebase pairs have disassociated. The equation for calculating the Tm. ofnucleic acids is well known in the art.

[0041] Two DNA sequences are “substantially homologous” when at leastabout 75% (preferably at least about 80%, and most preferably at leastabout 90 or 95%) of the nucleotides match over the defined length of theDNA sequences. Sequences that are substantially homologous can beidentified by comparing the sequences using standard software availablein sequence data bands, or in a Southern hybridization experiment under,for example, stringent conditions as defined for that particular system.Suitable conditions include those characterized by a hybridizationbuffer comprising 0.9M sodium citrate (“SSC”) buffer at a temperature ofabout 37° C. and washing in SSC buffer at a temperature of about 37° C.;and preferably in a hybridization buffer comprising 20% formamide in0.9M SSC buffer at a temperature of about 42° C. and washing in 0.2×SSCbuffer at about 42° C. Stringency conditions can be further varied bymodifying the temperature and/or salt content of the buffer, or bymodifying the length of the hybridization probe as is known to those ofskill in the art. Defining appropriate hybridization conditions iswithin the skill of the art. See e.g., Sambrook, J. Fritsch, E. J., &Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Lab. Press, Plainview, N.Y.).

[0042] The term “probe” as used herein refers to a labeledoligonucleotide which forms a duplex structure with a sequence inanother nucleic acid, due to complementarity of at least one sequence inthe probe with a sequence in the other nucleic acid.

[0043] The term “label” as used herein refers to any atom or moleculewhich can be used to provide a detectable (preferably quantifiable)signal, and which can be attached to a nucleic acid or protein. Labelsmay provide signals detectable by fluorescence, radioactivity,colorimetry, gravimetry, X-ray diffraction or absorption, magnetism,enzymatic activity, and the like.

[0044] The terms “nucleic acid substrate” and nucleic acid template” areused herein interchangeably and refer to a nucleic acid molecule whichmay comprise single- or double-stranded DNA or RNA.

[0045] “Oligonucleotide primers matching or complementary to a genesequence” refers to oligonucleotide primers capable of facilitating thetemplate-dependent synthesis of single or double-stranded nucleic acids.Oligonucleotide primers matching or complementary to a gene sequence maybe used in PCRs, RT-PCRs and the like.

[0046] A “consensus gene sequence” refers to a gene sequence which isderived by comparison of two or more gene sequences and which describesthe nucleotides most often present in a given segment of the genes; theconsensus sequence is the canonical sequence.

[0047] The term “native thrombomodulin” refers to both the naturalprotein and soluble peptides having the same characteristic biologicalactivity of membrane-bound or detergent solubilized (natural)thrombomodulin. These soluble peptides are also referred to as“wild-type” or “non-mutant” analog peptides. Biological activity is theability to act as a receptor for thrombin, increase the activation ofprotein C, or other biological activity associated with nativethrombomodulin. Oxidation resistant TM analogs are these solublepeptides that in addition to being soluble contain a specificartificially induced mutation in their amino acid sequence.

[0048] The term “thrombomodulin variant” is a polypeptide that differsfrom a native thrombomodulin polypeptide in one or more substitutions,deletions, additions and/or insertions, such that the bioactivity of thenative thrombomodulin polypeptide is not substantially diminished orenhanced. In other words, the bioactivity of a thrombomodulin variantmay be enhanced or diminished by, less than 50%, and preferably lessthan 20%, relative to the native protein. Preferred variants includethose in which one or more portions, such as an N-terminal leadersequence or transmembrane domain, have been removed. Other preferredvariants include variants in which a small portion (e.g., 1-30 aminoacids, preferably 5-15 amino acids) has been removed from the—and/orC-terminal of the mature protein.

[0049] Preferably, a thrombomodulin variant contains conservativesubstitutions. A “conservative substitution” is one in which an aminoacid is substituted for another amino acid that has similar properties,such that one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. Amino acid substitutions may generally be madeon the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity and/or the amphipathic nature of theresidues. For example, negatively charged amino acids include asparticacid and glutamic acid; positively charged amino acids include lysineand arginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. A variant may also, or alternatively,contain nonconservative changes. In a preferred embodiment, variantpolypeptides differ from a native sequence by substitution, deletion oraddition of five amino acids or fewer. Variants may also (oralternatively) be modified by, for example, the deletion or addition ofamino acids that have minimal influence on the bioactivity, secondarystructure and hydropathic nature of the polypeptide.

[0050] Thrombomodulin variants preferably exhibit at least about 70%,more preferably at least about 90% and most preferably at least about95% sequence homology to the original thrombomodulin polypeptide.

[0051] A thrombomodulin variant also include a thrombomodulinpolypeptides that is modified from the original thrombomodulinpolypeptides by either natural processes, such as posttranslationalprocessing, or by chemical modification techniques which are well knownin the art. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature. Modifications can occur anywhere in a polypeptide, includingthe peptide backbone, the amino acid side-chains and the amino orcarboxyl termini. It will be appreciated that the same type ofmodification may be present in the same or varying degrees at severalsites in a given polypeptide. Also, a given polypeptide may contain manytypes of modifications. Polypeptides may be branched, for example, as aresult of ubiquitination, and they may be cyclic, with or withoutbranching. Cyclic, branched, and branched cyclic polypeptides may resultfrom posttranslation natural processes or may be made by syntheticmethods. Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross links, formationof cysteine, formation of pyroglutamate, formulation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

[0052] Adenovirus Vectors:

[0053] The genome of an adenovirus can be manipulated such that itencodes and expresses a gene product of interest but is inactivated interms of its ability to replicate in a normal lyric viral life cycle(Curie D T, Ann N Y Acad Sci 886, 158-171 [1991]). Suitable adenoidalvectors derived from the adenovirus strain Ad type 5d1324 or otherstrains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to thoseskilled in the art. Recombinant adenovirus es are advantageous in thatthey do not require dividing cells to be effective gene deliveryvehicles and can be used to infect a wide variety of cell types,including airway epithelium, endothelial cells and muscle cells.Additionally, introduced adenoidal DNA (and foreign DNA containedtherein) is not integrated into the genome of a host cell but remainsepisomal, thereby avoiding potential problems that can occur as a resultof insertional mutagenesis in situations where introduced DNA becomesintegrated into the host genome (e.g., retroviral DNA). Moreover, thecarrying capacity of the adenoidal genome for foreign DNA is large (upto 8 kilobases relative to other gene delivery vectors (Haj-Ahmand etal. J. Virol. 57, 267-273 [1986]). Most replication-defective adenoidalvectors currently in use are deleted for all or parts of the viral E1and E3 genes but retain as much as 80% of the adenoidal geneticmaterial. Adenoidal vectors deleted for all viral coding regions arealso described by Kochanek et al. and Chamberlain et al. (U.S. Pat. No.5,985,846 and U.S. Pat. No. 6,083,750).

[0054] Adenovirus vectors have been successfully tested in a number ofanimal models (Ragot et al. Nature 361, 647-650 [1993]; Howell et al.Hum Gene Ther 9, 629-634 [1998]). Nonetheless, the toxicity andimmunogenicity remain major hurdles to overcome before the adenovirusvectors can be safely used in humans.

[0055] Adenoviruses (Ad) are double-stranded DNA viruses with a lineargenome of about 36 kb. The adenovirus genome is complex and containsover 50 open reading frames (ORFs). These ORFs are overlapping and genesencoding one protein are often embedded within genes coding for other Adproteins. Expression of Ad genes is divided into an early and a latephase. The early genes comprise E1a, E1b, E2a, E2b, E3 and E4, which aretranscribed prior to replication of the viral genome. The late genes(e.g., L1-5) are transcribed after replication of the viral genome. Theproducts of the late genes are predominantly components of the virion,as well as proteins involved in the assembly of virions.

[0056] The so-called “gutless” rAd vectors contain a minimal amount ofadenovirus DNA and are incapable of expressing any adenovirus antigens(hence the term “gutless”). The gutless rAd vectors provide thesignificant advantage of accommodating large inserts of foreign DNAwhile completely eliminating the problem of expressing adenoviral genesthat result in an immunological response to viral proteins when agutless rAd vector is used in gene therapy. Methods for producinggutless rAd vectors have been described, for example, in U.S. Pat. No.5,981,225 to Kochanek et al., and U.S. Pat. Nos. 6,063,622 and 6,451,596to Chamberlain et al; Parks et al., PNAS 93:13565 (1996) and Lieber etal., J. Virol. 70:8944-8960 (1996).

[0057] The “inverted terminal repeats (ITRs) of adenovirus” are shortelements located at the 5′ and 3′ termini of the linear adenoviralgenome, respectively and are required for replication of the viral DNA.The left ITR is located between 1-130 bp in the Ad genome (also referredto as 0-0.5 mu). The right ITR is located from about 3,7500 bp to theend of the genome (also referred to as 99.5-100 mu). The two ITRs areinverted repeats of each other. For clarity, the left ITR or 5′ end isused to define the 5′ and 3′ ends of the ITRs. The 5′ end of the leftITR is located at the extreme 5′ end of the linear adenoviral genome;picturing the left ITR as an arrow extending from the 5′ end of thegenome, the tail of the 5′ ITR is located at mu 0 and the head of theleft ITR is located at about 0.5 mu (further the tail of the left ITR isreferred to as the 5′ end of the left ITR and the head of the left ITRis referred to as the 3′ end of the left ITR). The tail of the right or3′ ITR is located at mu 100 and the head of the right ITR is located atabout mu 99.5; the head of the right ITR is referred to as the 5′ end ofthe right ITR and the tail of the right ITR is referred to as the 3′ endof the right ITR. In the linear adenoviral genome, the ITRs face eachother with the head of each ITR pointing inward toward the bulk of thegenome. When arranged in a “tail to tail orientation” the tails of eachITR (which comprise the 5′ end of the left ITR and the 3′ end of theright ITR) are located in proximity to one another while the heads ofeach ITR are separated and face outward.

[0058] The “encapsidation signal of adenovirus” or “adenovirus packagingsequence” refers to the ψ sequence which comprises five (AI-AV)packaging signals and is required for encapsidation of the mature lineargenome; the packaging signals are located from about 194 to 358 bp inthe Ad genome (about 0.5-1.0 mu).

[0059] One aspect of the present invention relates to a viral backboneshuttle vector for the construction of gutless rAd vectors. In oneembodiment, the viral backbone shuttle vector of the present inventioncontains a left and a right inverted terminal repeats of adenovirus, anencapsidation signal (ψ) of adenovirus, a pBR322 replication origin, akanamycin resistance gene, and a stuffer sequence, which is thehypoxanthine phosphoribosyltransferase (HPRT) intron fragment with anapproximately 10 Kb. (FIG. 1 and SEQ ID NO:1).

[0060] The viral backbone shuttle vector of the present inventioncontains multiple restriction endonuclease sites for the insertion of aforeign DNA sequence of interest. In one embodiment, the viral backboneshuttle vector contains seven unique cloning sites where the foreign DNAsequence can be inserted by molecular cloning techniques that are wellknown in the DNA cloning art. The foreign DNA sequence of interesttypically comprises cDNA or genomic fragments that are of interest totransfer into mammalian cells. Foreign DNA sequence of interest mayinclude any naturally occurring or synthetic DNA sequence. The foreignDNA may be identical in sequence to naturally-occurring DNA or may bemutated relative to the naturally occurring sequence. The foreign DNAneed not be characterized as to sequence or function.

[0061] The size of foreign DNA that may be included in the shuttlevector will depend upon the size of the rest of the vector. Ifnecessary, the stuffer sequence may be removed to adapt large sizeforeign DNA fragment. The total size of foreign DNA may vary from 1 kbto 35 kb. Preferably, the total size of foreign DNA is from 15 kb to 35kb.

[0062] The foreign DNA may encode protein, or contain regulatory sites,including but not limited to, transcription factor binding sites,promoters, enhancers, silencers, ribosome binding sequences,recombination sites, origins of replication, sequences which regulateRNA stability and polyadenylation signals. The promoters used may varyin their nature, origin and properties. The choice of promoter dependsin fact on the desired use and on the gene of interest, in particular.Thus, the promoter may be constitutive or regulated, strong or weak,ubiquitous or tissue/cell-specific, or even specific of physiological orpathophysiological states (activity dependent on the state of celldifferentiation or the step in the cell cycle). The promoter may be ofeukaryotic, prokaryotic, viral, animal, plant, artificial or human,etc., origin. Specific examples of promoters are the promoters of thegenes PGK, TK, GH, α-EF1, APO, CMV, RSV etc. or artificial promoters,such as those for p53, E2F or cAMP.

[0063] In one embodiment, the viral backbone shuttle vector of thepresent invention comprises at least 15 contiguous bases of SEQ ID NO:1,preferably comprises at least 90 contiguous bases of SEQ ID NO:1, morepreferably comprises at least 300 contiguous bases of SEQ ID NO:1, andmost preferably comprises 3000 or more contiguous bases of SEQ ID NO:1.

[0064] The present invention also relates to a gutless adenoviral vectorthat carries the a DNA sequence encoding a native TM protein or avariant of a TM protein. In one embodiment, the DNA sequence iscontrolled by a constitutive promoter such as the CMV promoter or RSVpromoter. In another embodiment, the DNA sequence is controlled by aregulatable expression system. Systems to regulate expression oftherapeutic genes have been developed and incorporated into the currentviral gene delivery vectors. These systems are briefly described below:

[0065] Tet-onloffsystem. The Tet-system is based on two regulatoryelements derived from the tetracycline-resistance operon of the E. coliTn 10 transposon: the tet repressor protein (TetR) and the Tet operatorDNA sequence (tetO) to which TetR binds. The system consists of twocomponents, a “regulator” and a “reporter” plasmid. The “regulator”plasmid encodes a hybrid protein containing a mutated Tet repression(tetr) fused to the VP 16 activation domain of herpes simplex virus. The“reporter” plasmid contains a tet-responsive element (TRE), whichcontrols the “reporter” gene of choice. The tetr-VP 16 fusion proteincan only bind to the TRE, therefore activate the transcription of the“reporter” gene, in the presence of tetracycline. The system has beenincorporated into a number of viral vectors including retrovirus,adenovirus (Gossen and Bujard, PNAS USA 89: 5547-5551, [1992]; Gossen etal., Science 268: 1766-1769, [1995]; Kistner et al., PNAS USA 93:10933-10938, [1996]).

[0066] Ecdysone system. The Ecdysone system is based on the moltinginduction system found in Drosophila, but modified for inducibleexpression in mammalian cells. The system uses an analog of thedrosophila steroid hormone ecdysone, muristerone A, to activateexpression of the gene of interest via a heterodimeric nuclear receptor.Expression levels have been reported to exceed 200-fold over basallevels with no effect on mammalian cell physiology (No et al., PNAS USA93: 3346-3351, [1996]).

[0067] Progesterone-system. The progesterone receptor is normallystimulated to bind to a specific DNA sequence and to activatetranscription through an interaction with its hormone ligand.Conversely, the progesterone antagonist mifepristone (RU486) is able toblock hormone-induced nuclear transport and subsequent DNA binding. Amutant form of the progesterone receptor that can be stimulated to bindthrough an interaction with RU486 has been generated. To generate aspecific, regulatable transcription factor, the RU486-binding domain ofthe progesterone receptor has been fused to the DNA-binding domain ofthe yeast transcription factor GAL4 and the transactivation domain ofthe HSV protein VP16. The chimeric factor is inactive in the absence ofRU486. The addition of hormone, however, induces a conformational changein the chimeric protein, and this change allows binding to aGAL4-binding site and the activation of transcription from promoterscontaining the GAL4-binding site (Wang et al., PNAS USA 93: 8180-8184,[1994]; Wang et al., Nat. Biotech 15: 239-243, [1997]).

[0068] Rapamycin-system. Immunosuppressive agents, such as FK506 andrapamycin, act by binding to specific cellular proteins and facilitatingtheir dimerization. For example, the binding of rapamycin toFK506-binding protein (FKBP) results in its heterodimerization withanother rapamycin binding protein FRAP, which can be reversed by removalof the drug. The ability to bring two proteins together by addition of adrug potentiates the regulation of a number of biological processes,including transcription. A chimeric DNA-binding domain has been fused tothe FKBP, which enables binding of the fusion protein to a specificDNA-binding sequence. A transcriptional activation domain also has beenJ.″used to FRAP. When these two fusion proteins are co-expressed in thesame cell, a fully functional transcription factor can be formed byheterodimerization mediated by addition of rapamycin. The dimerizedchimeric transcription factor can then bind to a synthetic promotersequence containing copies of the synthetic DNA-binding sequence. Thissystem has been successfully integrated into adenoviral vectors.Long-term regulatable gene expression has been achieved in both mice andbaboons (Magari et al., J. Clin. Invest. 100: 2865-2872, [1997]; Ye etal., Science 283:88-91, [1999]).

[0069] Ex Vivo and In Vivo Thrombomodulin Gene Transfer

[0070] The instant invention uses a gutless adenovirus vector to expressa native thrombomodulin protein or a variant of the thrombomodulinprotein at a vessel graft or angioplasty site to prevent or reducere-occlusion and intimal hyperplasia. The amino acid sequence of humanthrombomodulin (SEQ ID NO: 2) and the DNA sequence encoding humanthrombomodulin (SEQ BD NO:3) have been reported (Suzuki et al. EMBO J.6:1891-1897, [1987]).

[0071] In one embodiment, the in vivo expression of thrombomodulin or athrombomodulin variant is used for the treatment of atheroscleroticcardiovascular disease (CVD). Though venous grafts can be used forbypass surgeries, the veins eventually, become occluded by thrombosisresulting the recurrence of the diseases. In this embodiment, TM genedelivery is used in coronary artery bypass grafting, and vascular graftprostheses to block thrombosis. Specifically, TM gene is introduced intoa segment of blood vessel in vitro using a gene transfer vector.

[0072] TM gene delivery can be also used for the reduction of no-intimaformation, for the prevention of atherosclerosis; for the prevention ofmyocardial infarction and for the inhibition of fibrinolysis inhemophilic plasma. TM gene transfer at the site of thrombus formation ispotent approach to reverse these vascular diseases.

[0073] In another embodiment, in vivo TM expression is achieved byembedding a gene transfer vector in a stent which is placed at thetreatment site following percutaneous transluminal coronary angioplasty,peripheral artery angioplasty, thrombectomy, or an intravascularstenting procedure.

[0074] In another embodiment, the in vivo expression of thrombomodulin,or a thrombomodulin variant is used for the treatment of end stage renalfailure (ESRD). ESRD patients often exhibit decreased antithromboticactivity due to low TM levels. In such patients, enhanced in vivo TMgene expression can be potentially very useful.

EXAMPLE 1 Construction of Gutless Viral Backbone Shuttle Vector

[0075] An embodiment of a gutless viral backbone shuttle vector pShuttleis shown in FIG. 1. Sequence portion containing R-ITR, PBR322 ori, Kan,L-ITR, and encapsidation signal was obtained from the pAdEasy systemfrom Stratagene. At bp 3667 of the original pShuttle sequence, there isa BamHI site just beyond the R-ITR. PCR primers were designed to includethe BamHI site and then was to create an EcoRI site at the end of theR-ITR. The R-ITR was PCR replicated and then digested with BamHI andEcoRI to create sticky ends. The viral backbone was then cut with bothBamHI and EcoRI. The BamHI cut the backbone at bp 3667 and there wasalso an EcoRI site inside the MCS at bp 377. The backbone portion of theplasmid was then gel purified and the PCR replicated R-ITR was reclonedinto position. This essentially puts the L-ITR, encapsidation signal,MCS, and R-ITR all in close proximity to each other.

[0076] Insertion of the HPRT introns was a two step cloning process.First, the viral backbone was digested with EcoRI and XbaI, both enzymesites are in the MCS. The HPRT source was also digested with EcoRI andXbaI yielding a 7477 bp fragment that was cloned into the EcoRI/XbaIdigested viral backbone. Then the HPRT source was digested with onlyXbaI yielding a 2715 bp fragment. One of the XbaI sites in this cut isthe same XbaI site that was cut from the EcoRI/XbaI double digest instep 1. The viral backbone was cut with only XbaI and the 2715 bpfragment was inserted.

[0077] Overall, from the HPRT source, the HPRT stuffer sequence isinserted into the viral backbone in reverse orientation, hence intron 5,then 4, then 3. The 2715 bp fragment was inserted and checked to followthe original source sequence.

EXAMPLE 2 . Construction and Preparation of Gutless Viral Shuttle Vector

[0078] (a). Construction and Preparation of Gutless Viral Shuttle VectorCarrying Human Thrombomodulin Gene

[0079] The insertion of hTM into the gutless adenovirus backbone firstrequired the creation of a CMV-HTM expression cassette.

[0080] The intermediate vector used was pcDNA3.1I/Zeo(+) (Invitrogen). ACMV promoter is available commercially and a CMV promoter was clonedinto the multiple cloning site (MCS) at the XbaI/EcoRV restrictionenzyme site locations. The CMV from ps5 was removed using XbaI/EcoRV.pcDNA3.1/Zeo(+) was cleaved inside the MCS using both XbaI and EcoRV aswell. The CMV promoter was then ligated. Due to the location of theenzyme sites in the MCS, the CMV promoter (FIG. 6, SEQ ID NO:5) wasinserted in a backwards orientation relative to the pcDNA3.1/Zeo(+)plasmid. The TM cDNA (FIG. 7, SEQ ID NO:6) was obtained from Dr. Sadler(Dittman et al., Biochemistry, 26(14):4350-4357 [1987]) which thesequence was also submitted to ATCC and to GenBank. The TM gene wasremoved from the plasmid using EcoRI and inserted into pcDNA3.1/Zeo(+),also in the reverse orientation to pcDNA3.1/Zeo(+) downstream of theinserted CMV promoter. To remove the cassette, PmeI enzyme was used tocut both ends of the MCS. The gutless adenovirus backbone was linearizedusing SmaI which is at bp 381 of the backbone. The two were ligatedtogether in the forwards orientation with respect to the gutless virusbackbone. Sequence of the expression cassette (from PmeI site to PmeIsite, SEQ ID NO:4) is shown in FIG. 5.

[0081] (b). Construction and preparation of gutless viral shuttle vectorcarrying LacZ gene The insertion of LacZ also required creation of anintermediate vector to create the expression cassette. pcDNA3.1/Zeo(+)was again used. First, a portion of the vector from the end of the MCS,restriction enzyme site ApaI, to the beginning of the SV40 poly A,restriction site Nael, was removed and the vector relegated to itself.Then the LacZ gene was inserted into the vector MCS using NotI/XbaI. Theexpression cassette, containing CMV promoter, LacZ gene, and SV40 polyA, was removed using NruI/SalI retraction enzymes and blunt-end clonedinto the gutless adenovirus at the SmaI restriction enzyme site.

EXAMPLE 3 Preparation of Gutless Adenovirus

[0082] The helper virus is an E1/E3 deleted adenovirus in which aspecial flp recognition sequence site (FRS) flanks the encapsidationsignal. Helper adenovirus need to be grown in 293 cells.

[0083] 293 cell line has long ago been engineered to express E1 and E3genes of adenovirus. These two genes are necessary for viralreproduction. The flp gene is similar in function to Cre-Lox. The flpgene will recognize the FRS, cleave at that location, and then relegatethe DNA. It's basic function is to promote recombination betweendifferent pieces of DNA with the FRS, but in this case, it will cleaveout the encapsidation signal thereby not allowing helper-viral DNA to bepackaged. (Beauchamp et al., Molecular Therapy, 3(5):809-815 [2001];Umana et al., Nature Biotechnology, 19:582-585 [2001])

[0084] 293-flp cells will be transfected with the backbone DNA usingLipofectamine. While performing the transfection, helper virus were alsoused to infect the 293-flp cells. The helper virus were inserted its ownDNA into the 293-flp cells. The flp protein expressed in the cells willcleave the encapsidation signal thereby not allowing the helper virusDNA to package. Consequently the gutless adenovirus backbone DNA waspackaged into the adenoviral proteins expressed from the helper virusDNA. This virus will not be able to replicate in normal cells due to theE1/E3 deletions and will also contain the TM expression cassette.

[0085] The virus were produced by the following procedure:

[0086] (a) Virus Reproduction

[0087] Seed 293 cells in 15 cm dishes and grow in 10% FBS untilapproximately 70% confluent. Viral media was made as follows. 2 mL ofFBS-free IMEM containing antibiotic, antimycotic; adjust pfu per cell ofpurified virus until reached the final concentration of media as 1 μlvirus in 2 ml IMEM (viral Conc. 1×10¹⁰ pfu/mL)/each 15 cm Dish. ForExample: 30 Dishes=60 mL IMEM+30 μl virus Old media was Aspirated fromdishes, and 2 ml viral media was added per dish. Dishes were rocked at37° C. for 1.5-2 hours, and 18 mL 10% media was added per dish andincubated according to time course.

[0088] Cells were harvested by pipeting and collocating in 50 mL tubesat 4° C., and cells were centrifuge at 4° C., 2000 rpm for 5 min. Save10 mL of supernatant from one of the tubes into a separate tube. Thesupernatant was removed from all of the tubes. Take 5 mL supernatantfrom the saved tube and resuspend all the pellets to one tube. All ofthe tubes were re-wash with the remaining 5 mL of supernatant to collectany leftover sample, and the pellet was store at −80° C.

[0089] (b) Virus Collection

[0090] Sample tube(s) were frozen/thawed 5 times to lyse the cells, andthe virus were released using dry ice and incubated at 37° C. water bathfor 15 minutes until each to obtain crude viral lysate (CVL). The CVLwas collected in two 2059 Falcon Tubes and centrifuged using Sorvall HS4at 7000 rpm, 4° C. for 5 minutes and the supernatant was recovered.

[0091] To purify the virus, ultra-clear SW41 (Beckman) tubes wereprepared by soaking in Ultra Pure Water, then 70% ETOH. Cotton swabs(one swab for each tube) was used to completely dry out the tube, andtwo tubes were used per sample.

[0092] Preparation of the first gradient: 2.5 mL CsCl—Density 1.25, and2.5 mL CsCl—Density 1.40. Place the 1.25 density CsCl into the Beckmantubes first. Underlay slowly the high density, 1.40 CsCl using a sterilepasteur pipette, and overlay an equal amount (in mL) of CVL, about 4.25ml/tube. Samples were centrifuged in a SW41 rotor with speed: 35,000 rpmat 20° C. for 1 hour and with acceleration: 1 and deceleration: 4. Thelower opalescent band was collected using 1 or 3 mL syringe with greencap needles.

[0093] Preparation of second gradient: CsCl was prepared to density1.33. Two fresh ultra-clear tubes were placed 8 mL of CsCl and overlaythe band just recovered after the first spin. (To equilibrate the tubes,measure before the volume of the recovered band and divide equally inthe 2 tubes). Samples were centrifuged at the conditions above for 18hours. The opalescent band was recovered and collected in a sterileeppendorf tube. (From this moment, keep the tube always on ice). Sampleswere dialyze with dialysis buffer: (1) 10× Dialysis Buffer: 100 mMTris—pH 7.4, 10 mM MgCl₂; (2) 1× Dialysis Buffer (2 Liters): 400 mLGlycerol,200 mL 10× Dialysis Buffer 140 mL, and Ultra Pure Water. Thedialyzed samples were immediately stored at −70° C.

[0094] (c) Determination of Virus Titer

[0095] BioRad protein estimation kit was used with 1:5 diluting, andplacing 1 ml in each disposable cuvette. Standards were set up at 0, 1,2, 5 10, and 15 μg/ml. (BSA is fine). Sample cuvettes were preparedusing 1-10 μl of sample, depending on estimate of titer. (Sample OD mustbe within the linear range of the standard line.) OD was taken at 595%and formula of the line was calculate from standards. The proteinconcentration of the samples were calculated using this formula. Thefollowing formula was used to convert protein concentration to titer:[12.956+224.15 (μg/ml)]×10⁸.

EXAMPLE 4 Expression of Human Thrombomodulin (hTM) In Vitro

[0096] When enough hTM gutless adenovirus has been produced, experimentswill be performed to demonstrate the viable expression of hTM in HUVECcells post infection with the hTM containing gutless adenovirus. Todetect hTM expression post infection of HUVEC cells, RT-PCR will beperformed using hTM specialized primers to detect for thrombomodulinmRNA. Also, western blots will be performed to detect hTM proteinexpression by the HUVEC cells.

[0097] As a control, the same HUVEC cells will be infected the gutlessadenovirus expressing LacZ. These cells will subsequently be stainedwith X-gal to look for blue cells. This will demonstrate the viabilityof the gutless adenovirus backbone itself.

EXAMPLE 5 Composition of the Complete Viral Delivery System (CVDS)

[0098] The Complete Viral Delivery System composes of 1:1 mixture ofHam's F12 medium and DMEM, an effective amount of a gutless virus vectorcarrying a polynucleotide encoding a thrombomodulin protein or a variantof a thrombomodulin protein, and an acellular oxygen carrier. Preferredoxygen carrier includes: unmodified or chemically modified hemoglobin inthe range of 3 g/dl to 10 g/dl and perfluorochemical emulsions. The CVDSmay optionally contain 1 mM L-glutamine (Sigma), 1.5 g/L sodiumbicarbonate (Sigma), 1× antibiotic-antimycotic (Gibco 15240), and. TheCVDM maintains tissue viability during the viral treatment of bloodvessel.

EXAMPLE 6 Ex Vivo Treatment of Cardiovascular Disease

[0099] A vein segment is harvested from the leg and is stored in Ham'sF12 medium. Gutless adenovirus suspended in CVDM is then injected intothe isolated vein segment and incubated for 10 to 40 minutes dependingon the desired level of transfection. The infection may be performedunder pressure to enhance efficiency.

[0100] After the incubation, the vein segment is washed several times toeliminate all viral particles that have not entered the endothelialcells of the vein segment, and is then grafted into the desiredtreatment site. The thorough rinse avoids the spread of the viral vectorto other organs of the body following in situ grafting, and any systemicimmune response to the viral vector.

EXAMPLE 7 In Vivo Treatment for Peripheral Vascular Disease

[0101] In this application, the vein in the leg is treated followingevacuation of the clot. A catheter is inserted in the leg vein and boththe proximal and distal balloons are inflated to isolate the veinsegment to be transfected. The segment is evacuated of all blood, rinsedwith physiologic saline. The segment is then filled with the CVDSdescribed above, under pressure. The isolated vein segment is exposed tothe CVDS for a period of 10 to 45 minutes, depending upon the desiredtransfection efficiency.

EXAMPLE 8 In Vivo Treatment for Renal Disease

[0102] In this application, the vein in the kidney is treated followingevacuation of the clot. A catheter is inserted in the kidney vein andboth the proximal and distal balloons are inflated to isolate the veinsegment to be transfected. The segment is evacuated of all blood, rinsedwith physiologic saline; it is then filled with the CVDS describedabove, under pressure. The isolated vein segment is exposed to the CVDSfor a period of 10 to 45 minutes, depending upon the desiredtransfection efficiency.

EXAMPLE 9 In Vivo Treatment with Virus Containing Stent

[0103] In this application, a virus-coated stent is placed at atreatment site after angioplasty. The virus is a gutless adenoviruscarrying a polynucleotide encoding a thrombomodulin protein or a variantof a thrombomodulin protein. Alternatively, the virus may be embedded inthe stent and is releases gradually through a time-releasing mechanismwell-known to one skilled in the art.

[0104] The above description is for the purpose of teaching the personof ordinary skill in the art how to practice the present invention, andit is not intended to detail all those obvious modifications andvariations of it which will become apparent to the skilled worker uponreading the description. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence whichis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

1 6 1 13600 DNA Artificial Sequence gutless backbone shuttle vector 1catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 60ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 120gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 300agtgaaatct gaataatttt gtgttactca tagcgcgtaa tactggtacc gcggccgcct 360cgagtctaga actagtggat cccccggctg caggaattct gatggctctc aaaattcctg 420cctcctttag gggataaaag actttaagac tttttaacaa aaaagaaaaa gaaaaaaaaa 480attcctgcct cctggtgtac acacacagaa gggttccctc cccttgaatg tgaccaggat 540ctgtgaaaat aacgggatag ccgctcctgt gattaggtta tgtggtagac tagagcaaga 600ttctcctgct ggttttgaag aagtcagctg ccatgttgtg agactgtcat ggggctaggg 660catgagcctt ttaaatatct gggagcaacc cctggccagc agccagtgag aaaacgggcc 720ctcagtccta caatcacaag gaactaaatt ctgccaacaa cctgaaggaa ctttgaagag 780gatcatgagt cccttgattc agcttgatga gcccctgagc agaggataca gctaacttgt 840actagggaag tataaaaaac atgcatggga atgatatata tcaactttaa ggataattgt 900catacttctg ggaatgaagg gaaagaaatg gggctttagt tgtattatga tctttaattt 960ctcaaaaaaa agatcagaag caaatatggc aaaatgttaa tacttttgtg ggtacgtagg 1020tattcagcat accctttttt ctgagttcaa aatattttat aattaaaatg aaatgcaggc 1080caggcacagt ggctcatgcc tataatacca gcactttgcg aggccgaggt gggaggatgg 1140cttgaggcca gaccagcctg gccaacatgg caaaacccca tctctactta aaaaaaaaaa 1200aactatatat atatatatgt gtgtgtgtgt gtatatatat atatgtatat atatttatat 1260atgtgtgtat atatatatat gtatatatat ttatatatgt gtgtgtatat atatatatac 1320acacacacac atatatacat acatacatac acacacacac acacacaatt agccaggcat 1380ggtggcgcac acctgtagtc ccagctactt gggaggctga gacatgagaa ttgcttgaac 1440ctgggaggca gagtagttag tgagctgaga tcataccact gcactccagc ctggtgacag 1500agtgagactc tgtcttaaaa aaaataaaaa ttaaaattaa atgcaaaagg tccaagtgaa 1560ttgaagagga aaggggtatc aaggaaggtt ttgtggaggt gacgtttgag ctgggtctta 1620aatgacttaa acatgggata agaagggagg gaataaggac atttcaggta cgagaaataa 1680ggagcatcag tggaaacaac ctaacgtctg tcaaccagtg aatggataac aaaaatgtaa 1740ttcagatggt atccaactta cgatggttcc aacatgagat ttttctgact ttaggataga 1800tttatcaaag tagtaaatcc attttcaact tatgatattt tcaacttcag atgggtttat 1860caggacacag ttgaggaaca cctgtctatc catacaattt ggcaataaaa aggaaatgag 1920tgcagatata ctccacaaca tgaatgaacc ttgaaaacat taagtgagag aagccagata 1980caaaaggcca catattgtat gattctattt atacaaaatg tccagaatag gcaaatctta 2040tagacagcaa gtaggtagat gatcagtttg ctaggtgctg ggggaagggg aaatggggag 2100tgatggctaa ggggattggg tttctttgtg gggaaatgaa aatgttttaa aattgagcgt 2160gataatgatt gctaatgctg catatatata taatctatag attatatata tataaagaga 2220ggctgttaga cagtgataag tgatatatat atatatatac ataagagaga gagagagaga 2280gagagagagg ctgttagtga taagtgatca ggaaaataaa agtattgagg aggaatacga 2340agttgacggt gtgaaaacat gagattttat ataggatggc cagggaggcc ttaatgagaa 2400agtgacttat gagtaaaaac aagggatcct aaaccttagc atgcatcaga atcactcgga 2460aacttgttaa agcatagctt gctgggcctc atcacagata ttttgattcg gtaggttctt 2520gtctgatatt aatacttttg gtctagggaa ccacattttg agaaccactg agctaaagga 2580agtaaaggtt tcccttagtt tactagctgg taaccctagg aaactgctta gcctctcggt 2640gctaagatac aaaatacttt agcacataat aacacatgga aaatagtcta taaattataa 2700atattatttt ttatgtacca aatattacat aagacaaaat ctaagcaagt atatatatat 2760atacataaaa tataagatat atatgtatat attatatata gataaataga gagagagagt 2820tatgtttaga aagaaaatac ttcaaactaa aaaaagagag gtaggaagta taccattcca 2880ttattggtaa aaacaaatta ctaagtagtc tttacaaaaa acaatctcac tcctttagaa 2940cacaagccca ccattaaaac tgatgcagag gaatttctct ccctggctta cctttaggat 3000ggtgcatact aagttagaaa agtcataaat gttatattaa aagtaaatgt gaacttactt 3060ccacaatcaa gacattctag aagaaaaaga gaaatgaaaa tcagtacaat gaataaaacg 3120gtatttccaa ttataagtca aatcacatca taacaaccct aaggaattat ccaaactctt 3180gtttttagat gctttattat atcaaactct cctttaaaca agtggcccat ctgctgggat 3240ttggaagcct gtaatactga aattttcatc ataatggaaa ttttaaaaac agaattgacc 3300cacctgtttt taaaacactt tcattactta acaagaggtc taatcttggg caagtcttga 3360aatttctctg gccttagttt cccatgtgtt aaatgaaact tgaagcagtt ggtctcttat 3420agtctcctga ctctaacatt ctaagaatta tatttgtaca ataactcaaa aatcacataa 3480tttaatttac catatggact ccaaaatata ttttctcatt aggctaaact tgatctgcat 3540tttctggatg tgtccatatt cttggactac actaaaacat gataccaatg cttcctctca 3600ccataaaccc tcacttcgct ttctacattt aagaatttta tagctggaag agtccttaag 3660agaaaatacc atctaataat tacccctcaa aatcgagaaa gtcctatctg ttcttatgct 3720agttataaga atgaggcagc atttcacata atggttataa acactgccac aagaagattc 3780atgatgtgtt gtttatctgt agctctcatc atactctgtc atataactat agcattaaga 3840ttttaatgtt ctatatattc ttctaagaca gtgtttacca gagtaaggca caaaagatcc 3900actggtttgc aagaaagatt agaactttta aattttttaa cctcaccttg tttaatctat 3960atttttgtat gtattttgta acatatatat tattattacc ataaatcata tataatttaa 4020aatgcatata ttaggggtaa atgctcagga aactttttat aaattgggca tgcaaataca 4080agtttgaaga ctcactgttc taggtattaa aagtaaagtt ataaccaagt aaagcttcca 4140ccttttcatg tctcaaagca gtttattgtt ggaggtaaga tctcttagaa gcctaaacag 4200gtccaagtac agaatgaagt aaggctagcc cataacttgt ggcaagcaat tcatactatt 4260tctctcatgc tgagctctcc tcagtgaagc agctactata gacaactgca gcctattggt 4320agcctatttt acaggcagga aaaaaattac ttttttattc aaagtggaac tcaggacatg 4380gggagaaaat gaatacaaaa aatagggtca atccaaaggc acacagcaaa tgagtaacac 4440agttatgttt ttttcccatt tgtatgaggt cccagtaaat tctaagtaaa ctgcaaattt 4500aataatacac taaaaaagcc atgcaattgt tcaaatgaat cccagcatgg tacaaggagt 4560acagacacta gagtctaaaa aacaaaagaa tgccattatt gagtttttga attatatcaa 4620gtagttacat ctctacttaa taaatgagaa aaacgaggat aagaggccat ttgataaaat 4680gaaaatagcc aagaagtggt attagagact tgaatacagg tattcgggtc caaagttcat 4740ctgctcaaat actaactggg gaaaagaggg aaaaatattt atatacatat atatctgcac 4800aaaaataccc ccaaaagaca aaatgaggcc aggcagggtg gctcacaccc gtaatcccgg 4860tactttggga ggctgaggca ggtggatacc tgagatcagg agttggagat cagcctggtc 4920aacatggtga aaccctgtct ctactaaaag ataaaaaaat tagccaggca tggtggcgtg 4980cgcctgtaat cccagctact tgggagtctg aggcaggaga atcacttgaa ctgggaaggg 5040gaggttgcag tgagccaaga tcgtactact gcactccagc ctgggcagca gagtgagact 5100ccatcacaaa ataaataaat aaataaaata caatgaaaca gaaagttcaa ataatcccat 5160aatcttacca ccaagaaata actttcactc gttatactta ttgatttttc cataataaat 5220gtactttact gtgactatca tgaaaagaaa gttattttag aaacagagaa ctgtttcaga 5280tcaaatctat gtagtagaac agagccatta ggtgggaaag acgagatcaa actaaatctc 5340agaaggccta aaaggctagg tccattccag cactaaaaac tgaccagaca agtaatggct 5400tcaacagctt ctaaatatgg acaaagcatg ctgaaaggga aggacaggtc taacagtggt 5460atatgaaatg aacaggaggg gcaaagctca tttctcctct gaagttttcc aaagatgctg 5520aggaggacat tagtttgaca tgaccctgat atgggacaag ataatttcac agaagtttta 5580catgttaaag ttttcttata gatactcatt caagtaagca atgaacacta aaatctaaag 5640aaagaaaaga gctttagagt caggtctgta ttcaaattca agctctacca cttactggtt 5700ctgtgacttt gggcaagtct tttaccctta ttaagtctta atttcctgat ttgtaaaatg 5760gggatatcgt ctccctcaca ggattgttgt gaaactttta tgagattaat gcctttatat 5820ttggcatagt gtaagtaaac aataactggc agcttcaaaa aaaaaaagca gtagcattcc 5880atcatttatt attggttact ctcaaaaagt ttttcaatgt actagaagat aaatattcaa 5940ataccttaat atctccatta ttttcaggta aacagcatgc tcctgaacaa ccaatgggtc 6000aacaaataaa ttaaaaggga aatctaaaaa catcttgata ttaaactaca tggaagcaca 6060atataccaaa accatggttc acactaggag aattttaagg tacaagaaaa ctctttgaga 6120tttcttaaaa taatagtatg tctgaattta ttgagtgatt taccagaaac tgttgtaaga 6180gctctacttg cattatagca cttaatcctc ttaactctat ggctgctatt atcaacctca 6240ccctaatcac atatgggaca cagagaggtt aagtaacttg cccaaggtca gagttaggaa 6300gtactaagcc atgctttgaa tcagttgtca ggctccggaa ctcacacttt cagccactac 6360ataatactgc tttgctatct tttaggaaac tatgtgagtc tacctcacat agactcacat 6420aggtttgttt tttttttttt tttaaaggct atcttttccc ccatcaatgt tttttgaagg 6480atcccaaatt agagtcccac agaggcagac agcagtactt gacaatatgg acatttaagg 6540ttaatgttgg attctactgt ctttttacta catgacctag ggaacgataa ttaacctaga 6600ctgcttccaa gggttaaata acccatttag ttatactatg taaattatct cttagtgatt 6660gattgaaagc acactgttac taattgactc ggtatgaagt gctttttttt cttccctttc 6720aagatacata cctttccagt taaagttgag agatcatctc caccaattac ttttatgtcc 6780cctgttgact ggtcattcta gttaaaaaaa aaaaaactat atatatatat atctacacac 6840acatatgtat atgtatatcc ttatgtacac acacaaactt caaattaaat gagaactaga 6900agatttgaga agttagctag ctaatatcca tagcattatg atattctaaa tgatatgaat 6960tataagaatt aggtttcctg aaatgaatga ctagaaaact ttcaagtaga gattagtaaa 7020aattaaaaag tcctaatcgg ccattactga tttgatgttt ttaagagtcc taaaaaatgg 7080gttacatcca tttttaagtg ggtagtatta taacagccac ccatcttcaa tcacagtgat 7140ttctgaattg tgagggaagt tattagcatg acaggtgtct ggttctggcc ctgtacgatt 7200cccatgagtc aagcaaattg taagggctgg tctatatcac acccaacccc aaggatatgt 7260ccctcaaaag tctagcccag gccccgtcat cttcagcatc atctgggaaa ccaggtctga 7320ttagtagtcc tttaaggata cctcttaggc tcccatttta ctgctatcac agaatccaat 7380aaaaccctta caggagattc aatgggaaat gctcaacacc cactgtagtt ggtggtgaca 7440atgaccataa tttggctgtg ctggattcag gacagaaaat ttgggtgaaa gagcaggtga 7500acaaaagagc ttcgacttgc cctagcagag agcaagccat accataccac aaagccacag 7560aattacaacg gtgcagtacc agcacagtaa atgaacaaag tagagcccag aaacagaccc 7620agaactatat gaggatttag tatacaataa agatggtatt tcgagtcagt agggaaaaga 7680tgaattattc aataaatgat gtttggccaa ctagtaaccc atttgggaaa aaataaaagt 7740atggtcccta cctcacagca tacacaaaaa taaattccag acggattaaa atctaaatgt 7800aaaaaataaa gccataagtg gactggaaga aaatagagaa ttttttttaa catccgtaga 7860aagggtaaaa acccaggcat gacatgaacc aaaactgaag aggttctgta acaaataccc 7920ccttttatat attgggctcc aacaataaga acccatagga aaatggagaa tgaacacaaa 7980tagacaattt atagaagaga aggttataag gtgtaaaatt atatctatct gagaaacaaa 8040cactaaaaca atgtgattct actgttctcc cacccatact ggcaaaactt aagcctgata 8100atatgctgag gggaaataag cactcttgtt ggtgagagta ttaattggca tagcttcttt 8160tgaaaatgac atagcaatac ctgttaaaat tgcaaacatg catgtcactt aattccatgt 8220aattcccact tctgggaatc aattgctaca aaaacacttg acaagtatac aaagatacat 8280tcaagagtgt tcactgggcc gggtgcggtg gcttcatgcc tgtaatccca gggaggcaga 8340ggcaagacga tcgcttgacc ccaggagttc aaggccagcc cgagaaacac agcaagaccc 8400tgtctctctt ttttttattt aaaaaataaa tgttcactgt atcagttgtt cacaaaaaca 8460aaccaacatg tccattaaca gggaaccatt taaattaatc aagttcatct acacaatgta 8520ataccatgca actattaaaa agcacctgat aatccaaagc acactgagac agaataatgc 8580tattaaaaac accaagtagt ggaacactgt gttgcctatg acaccatttt tattcaacat 8640ttaaacaaat ttgtaacagc aattacatga gtagtgacaa tggcgtttat gagacttttc 8700acttttatgt gcttctattt ttgttatgct tctatatata catccattta ttatggagtg 8760ttactttcaa aaatcacaaa tgggccagta ttatttggtg ttgcaaggtg agcatatgac 8820ttctgatatc aacctttgca tattacttct caatttaggg aaattacaga catcccttat 8880tctaactaac ttaaaaccca gcatttcaaa catacagaat tgatggggaa aaaaagaaag 8940aagaaagaaa gaaaaggcaa caagcttcag atgacagtga ctcacatcaa attatttata 9000aaatctgtta aatagtgcca tcttctggag atacctggta ttacagtcca actccagttg 9060atgtctttac agagacaaga ggaataaagg aaaaaatatt caagaactga aaagtatgga 9120gtcatggaaa aattgctgtg atccaaaggc tacggtgata ggacaagaaa caagagaact 9180ccaagcagta agacactgct gttctattag catccaaacc tccatacctc ctgtttgccc 9240caaggctttt ttaaaaaata gagacaggat ctcactattt tgctcaggct ggtcttgaac 9300tcctggactc aagctatcct cctgcctcgg cctcctaaag tgccgagatt acaggcttga 9360gtcaccatac ctggctattt attttttctt aactctcttg cctggcctat agccaccatg 9420gaagctaata aagaatatta atttaagagt aatggtatag ttcactacat tggaatacag 9480gtataagtgc ctacattgta catgaatggc atacatggat caattacccc acctgggtgg 9540ccaaaggaac tgcgcgaacc tccctccttg gctgtctgga acaagcttcc cactagatcc 9600ctttactgag tgcctccctc atctttaatt atggttaagt ctaggataac aggactggca 9660aaggtgaggg gaaagcttcc tccagagttg ctctaccctc tcctctaccg tcctattctc 9720ctcactcctc tcagccaagg agtccaatct gtcctgaact cagagcgtca ctgtcaacta 9780catcaaaatt gccagagaag ctctttggga ctacaaacac atacccttaa tgtctttatt 9840tctattttgt ctacctcttc agtctaggtg aaaaaatagg aaggataata gggaagaact 9900ttgtttatgc ctacttatcc gcccctagga attttgaaaa cctctaggta gcaataagaa 9960ctgcagcatg gtatagaaaa agaggaggaa agctgtatag aaatgcataa taaatgggca 10020ggaaaagaac tgcttggaac aaacagggag gttgaactat aaggagagaa agcagagagg 10080ctaatcaaca aggctgggtt cccaagaggg catgatgaga ctattactaa ggtaggaatt 10140actaagggct tccatgtccc cttagtggct tagtactatg tagcttgctt tctgcagtga 10200acttcagacc cttcttttag gatcctagaa tggacttttt ttttttatcg gaaaacagtc 10260attctctcaa cattcaagca ggccccaagt ctaccacact caatcacatt ttctcttcat 10320atcataatct ctcaaccatt ctctgtcctt ttaactgttt ttctataccc tgatcaaatg 10380ccaacaaaaa gtgagaatgt tagaatcatg tatttttaga ggtagactgt atctcagata 10440aaaaaaaagg ggcagatatt ccattttcca aaatatgtat gcagaaaaaa taagtatgaa 10500aggacatatg ctcaggtaac aagttaattt gtttacttgt attttatgaa ttccctaaaa 10560cctacgtcac ccgccccgtt cccacgcccc gcgccacgtc acaaactcca ccccctcatt 10620atcatattgg cttcaatcca aaataaggta tattattgat gatgttaatt aacatgcatg 10680gatccatatg cggtgtgaaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc 10740gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 10800tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 10860agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 10920cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 10980ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 11040tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 11100gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 11160gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 11220gtaactattc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 11280actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 11340tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 11400gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 11460ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 11520cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 11580ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 11640tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 11700agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 11760gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 11820ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 11880gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 11940cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 12000gcagccatga gattatcaaa aaggatcttc acctagatcc ttttcacgta gaaagccagt 12060ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg gacaagggaa 12120aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg atagctagac 12180tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggccct ctggtaaggt 12240tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct gatggcgcag 12300gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg 12360attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca 12420acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt 12480tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg 12540gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga 12600agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca 12660ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct 12720tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac 12780tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc 12840gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt 12900gacccatggc gatgcctgct tgccgaatat catggtggaa aatggcgctt ttctggattc 12960atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt 13020gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc 13080gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgaatt 13140ttgttaaaat ttttgttaaa tcagctcatt ttttaaccat aggccgaaat cggcaaaatc 13200ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag 13260agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc 13320gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaag 13380cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga 13440acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg 13500tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg 13560cgtccattcg ccattcagga tcgaattaat tcttaattaa 13600 2 574 PRT Homo sapien2 Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala Gly Leu Gly 1 5 1015 Phe Pro Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu 20 2530 His Asp Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala 35 4045 Ser Gln Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser 50 5560 Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly 65 7075 80 Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys 8590 95 Gly Asp Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr100 105 110 Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp LeuAsn 115 120 125 Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser AlaAla Glu 130 135 140 Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln GlnCys Glu Val 145 150 155 160 Lys Ala Asp Gly Phe Leu Cys Glu Phe His PhePro Ala Thr Cys Arg 165 170 175 Pro Leu Ala Val Glu Pro Gly Ala Ala AlaAla Ala Val Ser Ile Thr 180 185 190 Tyr Gly Thr Pro Phe Ala Ala Arg GlyAla Asp Phe Gln Ala Leu Pro 195 200 205 Val Gly Ser Ser Ala Ala Val AlaPro Leu Gly Leu Gln Leu Met Cys 210 215 220 Thr Ala Pro Pro Gly Ala ValGln Gly His Trp Ala Arg Glu Ala Pro 225 230 235 240 Gly Ala Trp Asp CysSer Val Glu Asn Gly Gly Cys Glu His Ala Cys 245 250 255 Asn Ala Ile ProGly Ala Arg Pro Cys Gln Cys Pro Ala Gly Ala Ala 260 265 270 Leu Gln AlaAsp Gly Arg Ser Cys Thr Ala Ser Thr Gln Ser Cys Asn 275 280 285 Asp LeuCys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser 290 295 300 TyrSer Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His 305 310 315320 Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro 325330 335 Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro340 345 350 Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp ProCys 355 360 365 Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn GlnThr Ser 370 375 380 Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile ProHis Glu Pro 385 390 395 400 His Arg Cys Gln Met Phe Cys Asn Gln Thr AlaCys Pro Ala Asp Cys 405 410 415 Asp Pro Asn Thr Gln Ala Ser Cys Glu CysPro Glu Gly Tyr Ile Leu 420 425 430 Asp Asp Gly Phe Ile Cys Thr Asp IleAsp Glu Cys Glu Asn Gly Gly 435 440 445 Phe Cys Ser Gly Val Cys His AsnLeu Pro Gly Thr Phe Glu Cys Ile 450 455 460 Cys Gly Pro Asp Ser Ala LeuAla Arg His Ile Gly Thr Asp Cys Asp 465 470 475 480 Ser Gly Lys Val AspGly Gly Asp Ser Gly Ser Gly Glu Pro Pro Pro 485 490 495 Ser Pro Thr ProGly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu Val 500 505 510 His Ser GlyLeu Leu Ile Gly Ile Ser Ile Ala Ser Leu Cys Leu Val 515 520 525 Val AlaLeu Leu Ala Leu Leu Cys His Leu Arg Lys Lys Gln Gly Ala 530 535 540 AlaArg Ala Lys Met Glu Tyr Lys Cys Ala Ala Pro Ser Lys Glu Val 545 550 555560 Val Leu Gln His Val Arg Thr Glu Arg Thr Pro Gln Arg Leu 565 570 31725 DNA Homo sapien 3 atgcttgggg tcctggtcct tggcgcgctg gccctggccggcctggggtt ccccgcaccc 60 gcagagccgc agccgggtgg cagccagtgc gtcgagcacgactgcttcgc gctctacccg 120 ggccccgcga ccttcctcaa tgccagtcag atctgcgacggactgcgggg ccacctaatg 180 acagtgcgct cctcggtggc tgccgatgtc atttccttgctactgaacgg cgacggcggc 240 gttggccgcc ggcgcctctg gatcggcctg cagctgccacccggctgcgg cgaccccaag 300 cgcctcgggc ccctgcgcgg cttccagtgg gttacgggagacaacaacac cagctatagc 360 aggtgggcac ggctcgacct caatggggct cccctctgcggcccgttgtg cgtcgctgtc 420 tccgctgctg aggccactgt gcccagcgag ccgatctgggaggagcagca gtgcgaagtg 480 aaggccgatg gcttcctctg cgagttccac ttcccagccacctgcaggcc actggctgtg 540 gagcccggcg ccgcggctgc cgccgtctcg atcacctacggcaccccgtt cgcggcccgc 600 ggagcggact tccaggcgct gccggtgggc agctccgccgcggtggctcc cctcggctta 660 cagctaatgt gcaccgcgcc gcccggagcg gtccaggggcactgggccag ggaggcgccg 720 ggcgcttggg actgcagcgt ggagaacggc ggctgcgagcacgcgtgcaa tgcgatccct 780 ggggctcccc gctgccagtg cccagccggc gccgccctgcaggcagacgg gcgctcctgc 840 accgcatccg cgacgcagtc ctgcaacgac ctctgcgagcacttctgcgt tcccaacccc 900 gaccagccgg gctcctactc gtgcatgtgc gagaccggctaccggctggc ggccgaccaa 960 caccggtgcg aggacgtgga tgactgcata ctggagcccagtccgtgtcc gcagcgctgt 1020 gtcaacacac agggtggctt cgagtgccac tgctaccctaactacgacct ggtggacggc 1080 gagtgtgtgg agcccgtgga cccgtgcttc agagccaactgcgagtacca gtgccagccc 1140 ctgaaccaaa ctagctacct ctgcgtctgc gccgagggcttcgcgcccat tccccacgag 1200 ccgcacaggt gccagatgtt ttgcaaccag actgcctgtccagccgactg cgaccccaac 1260 acccaggcta gctgtgagtg ccctgaaggc tacatcctggacgacggttt catctgcacg 1320 gacatcgacg agtgcgaaaa cggcggcttc tgctccggggtgtgccacaa cctccccggt 1380 accttcgagt gcatctgcgg gcccgactcg gcccttgcccgccacattgg caccgactgt 1440 gactccggca aggtggacgg tggcgacagc ggctctggcgagcccccgcc cagcccgacg 1500 cccggctcca ccttgactcc tccggccgtg gggctcgtgcattcgggctt gctcataggc 1560 atctccatcg cgagcctgtg cctggtggtg gcgcttttggcgctcctctg ccacctgcgc 1620 aagaagcagg gcgccgccag ggccaagatg gagtacaagtgcgcggcccc ttccaaggag 1680 gtagtgctgc agcacgtgcg gaccgagcgg acgccgcagagactc 1725 4 4454 DNA Homo sapien misc_feature 349 n = A,T,C or G 4gtttaaacgg gccctctaga cgcgttgaca ttgattattg actagttatt aatagtaatc 60aattacgggg tcattagttc atagcccatg atatcatatg gagttccgcg ttacataact 120tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat 180gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta 240tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc 300ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtnc atgaccttat 360gggactttcc tacttggcag acatctacgt attagtcatc gctattacca tggtgatgcg 420gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat tttccaagtc 480tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 540aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 600tctatataag cagagctctc tggctaacta gagaacccct gcttactggc ttatcgagat 660atctgcagaa ttcatctgtc gactgctacc ggcagcgcgc agcggcaaga agtgtctggg 720ctgggacgga caggagaggc tgtcgccatc ggcgtcctgt gcccctctgc tccggcacgg 780ccctgtcgca gtgcccgcgc tttccccggc gcctgcacgc ggcgcgcctg ggtaacatgc 840ttggggtcct ggtccttggc gcgctggccc tggccggcct ggggttcccc gcacccgcag 900agccgcagcc gggtggcagc cagtgcgtcg agcacgactg cttcgcgctc tacccgggcc 960ccgcgacctt cctcaatgcc agtcagatct gcgacggact gcggggccac ctaatgacag 1020tgcgctcctc ggtggctgcc gatgtcattt ccttgctact gaacggcgac ggcggcgttg 1080gccgccggcg cctctggatc ggcctgcagc tgccacccgg ctgcggcgac cccaagcgcc 1140tcgggcccct gcgcggcttc cagtgggtta cgggagacaa caacaccagc tatagcaggt 1200gggcacggct cgacctcaat ggggctcccc tctgcggccc gttgtgcgtc gctgtctccg 1260ctgctgaggc cactgtgccc agcgagccga tctgggagga gcagcagtgc gaagtgaagg 1320ccgatggctt cctctgcgag ttccacttcc cagccacctg caggccactg gctgtggagc 1380ccggcgccgc ggctgccgcc gtctcgatca cctacggcac cccgttcgcg gcccgcggag 1440cggacttcca ggcgctgccg gtgggcagct ccgccgcggt ggctcccctc ggcttacagc 1500taatgtgcac cgcgccgccc ggagcggtcc aggggcactg ggccagggag gcgccgggcg 1560cttgggactg cagcgtggag aacggcggct gcgagcacgc gtgcaatgcg atccctgggg 1620ctccccgctg ccagtgccca gccggcgccg ccctgcaggc agacgggcgc tcctgcaccg 1680catccgcgac gcagtcctgc aacgacctct gcgagcactt ctgcgttccc aaccccgacc 1740agccgggctc ctactcgtgc atgtgcgaga ccggctaccg gctggcggcc gaccaacacc 1800ggtgcgagga cgtggatgac tgcatactgg agcccagtcc gtgtccgcag cgctgtgtca 1860acacacaggg tggcttcgag tgccactgct accctaacta cgacctggtg gacggcgagt 1920gtgtggagcc cgtggacccg tgcttcagag ccaactgcga gtaccagtgc cagcccctga 1980accaaactag ctacctctgc gtctgcgccg agggcttcgc gcccattccc cacgagccgc 2040acaggtgcca gatgttttgc aaccagactg cctgtccagc cgactgcgac cccaacaccc 2100aggctagctg tgagtgccct gaaggctaca tcctggacga cggtttcatc tgcacggaca 2160tcgacgagtg cgaaaacggc ggcttctgct ccggggtgtg ccacaacctc cccggtacct 2220tcgagtgcat ctgcgggccc gactcggccc ttgcccgcca cattggcacc gactgtgact 2280ccggcaaggt ggacggtggc gacagcggct ctggcgagcc cccgcccagc ccgacgcccg 2340gctccacctt gactcctccg gccgtggggc tcgtgcattc gggcttgctc ataggcatct 2400ccatcgcgag cctgtgcctg gtggtggcgc ttttggcgct cctctgccac ctgcgcaaga 2460agcagggcgc cgccagggcc aagatggagt acaagtgcgc ggccccttcc aaggaggtag 2520tgctgcagca cgtgcggacc gagcggacgc cgcagagact ctgagcggcc tccgtccagg 2580agcctggctc cgtccaggag cctgtgcctc ctcaccccca gctttgctac caaagcacct 2640tagctggcat tacagctgga gaagaccctc cccgcacccc caagctgttt tcttctattc 2700catggctaac tggcgagggg gtgattagag ggaggagaat gagcctcggc ctcttccgtg 2760acgtcactgg accactgggc aatgatggca attttgtaac gaagacacag actgcgattt 2820gtcccaggtc ctcactaccg ggcgcaggag ggtgagcgtt attggtcggc agccttctgg 2880gcagaccttg acctcgtggg ctaggatgac taaaatattt atttttttta agtatttagg 2940tttttgtttg tttcctttgt tcttacctgt atgtctccag tatccacttt gcacagctct 3000ccggtctctc tctctctaca aactcccact tgtcatgtga caggtaaact atcttggtga 3060attttttttt cctagccctc tcacatttat gaagcaagcc ccacttattc cccattcttc 3120ctagttttct cctcccagga actgggccaa ctcacctgag tcaccctacc tgtgcctgac 3180cctacttctt ttgctcttag ctgtctgctc agacagaacc cctacatgaa acagaaacaa 3240aaacactaaa aataaaaatg gccatttgct ttttcaccag atttgctaat ttatcctgaa 3300atttcagatt cccagagcaa aataatttta aacaaaggtt gagatgtaaa aggtattaaa 3360ttgatgttgc tggactgtca tagaaattac acccaaagag gtatttatct ttacttttaa 3420acagtgagcc tgaattttgt tgctgttttg atttgtactg aaaaatggta attgttgcta 3480atcttcttat gcaatttcct tttttgttat tattacttat ttttgacagt gttgaaaatg 3540ttcagaaggt tgctctagat tgagagaaga gacaaacacc tcccaggaga cagttcaaga 3600aagcttcaaa ctgcatgatt catgccaatt agcaattgac tgtcactgtt ccttgtcact 3660ggtagaccaa aataaaacca gctctactgg tcttgtggaa ttgggagctt gggaatggat 3720cctggaggat gcccaattag ggcctagcct taatcaggtc ctcagagaat ttctaccatt 3780tcagagaggc cttttggaat gtggcccctg aacaagaatt ggaagctgcc ctgcccatgg 3840gagctggtta gaaatgcaga atcctaggct ccaccccatc cagttcatga gaatctatat 3900ttaacaagat ctgcaggggg tgtgtctgct cagtaatttg aggacaacca ttccagactg 3960cttccaattt tctggaatac atgaaatata gatcagttat aagtagcagg ccaagtcagg 4020ccttattttc aagaaactga ggaattttct ttgtgtagct ttgctctttg gtagaaaagg 4080ctaggtacac agctctagac actgccacac agggtctgca aggtctttgg ttcagctaag 4140ctaggaatga aatcctgctt cagtgtatgg aaataaatgt atcatagaaa tgtaactttt 4200gtaagacaaa ggttttcctc ttctattttg taaactcaaa atatttgtac atagttattt 4260atttattgga gataatctag aacacaggca aaatccttgc ttatgacatc acttgtacaa 4320aataaacaaa taacaatgtg aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4380ggtagcagtc gacagatgaa ttccaccaca ctggactagt ggatccgagc tcggtaccaa 4440gcttaagttt aaac 4454 5 649 DNA Homo sapien misc_feature 335 n = A,T,C orG 5 tctagacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 60tagttcatag cccatgatat catatggagt tccgcgttac ataacttacg gtaaatggcc 120cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca 180tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg 240cccacttggc agtacatcaa gtgtatcata tgccaagtac gcccccctat tgacgtcaat 300gacggtaaat ggcccgcctg gcattatgcc cagtncatga ccttatggga ctttcctact 360tggcagacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca 420tcaatgggcg tggatagcgg tttgactcac ggggattttc caagtctcca ccccattgac 480gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac 540tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga 600gctctctggc taactagaga acccctgctt actggcttat cgagatatc 649 6 3693 DNAHomo sapien 6 ggcagcgcgc agcggcaaga agtgtctggg ctgggacgga caggagaggctgtcgccatc 60 ggcgtcctgt gcccctctgc tccggcacgg ccctgtcgca gtgcccgcgctttccccggc 120 gcctgcacgc ggcgcgcctg ggtaacatgc ttggggtcct ggtccttggcgcgctggccc 180 tggccggcct ggggttcccc gcacccgcag agccgcagcc gggtggcagccagtgcgtcg 240 agcacgactg cttcgcgctc tacccgggcc ccgcgacctt cctcaatgccagtcagatct 300 gcgacggact gcggggccac ctaatgacag tgcgctcctc ggtggctgccgatgtcattt 360 ccttgctact gaacggcgac ggcggcgttg gccgccggcg cctctggatcggcctgcagc 420 tgccacccgg ctgcggcgac cccaagcgcc tcgggcccct gcgcggcttccagtgggtta 480 cgggagacaa caacaccagc tatagcaggt gggcacggct cgacctcaatggggctcccc 540 tctgcggccc gttgtgcgtc gctgtctccg ctgctgaggc cactgtgcccagcgagccga 600 tctgggagga gcagcagtgc gaagtgaagg ccgatggctt cctctgcgagttccacttcc 660 cagccacctg caggccactg gctgtggagc ccggcgccgc ggctgccgccgtctcgatca 720 cctacggcac cccgttcgcg gcccgcggag cggacttcca ggcgctgccggtgggcagct 780 ccgccgcggt ggctcccctc ggcttacagc taatgtgcac cgcgccgcccggagcggtcc 840 aggggcactg ggccagggag gcgccgggcg cttgggactg cagcgtggagaacggcggct 900 gcgagcacgc gtgcaatgcg atccctgggg ctccccgctg ccagtgcccagccggcgccg 960 ccctgcaggc agacgggcgc tcctgcaccg catccgcgac gcagtcctgcaacgacctct 1020 gcgagcactt ctgcgttccc aaccccgacc agccgggctc ctactcgtgcatgtgcgaga 1080 ccggctaccg gctggcggcc gaccaacacc ggtgcgagga cgtggatgactgcatactgg 1140 agcccagtcc gtgtccgcag cgctgtgtca acacacaggg tggcttcgagtgccactgct 1200 accctaacta acgacctggt ggacggcgag tgtgtggagc ccgtggacccgtgcttcaga 1260 gccaactgcg agtaccagtg ccagcccctg aaccaaacta gctacctctgcgtctgcgcc 1320 gagggcttcg cgcccattcc ccacgagccg cacaggtgcc agatgttttgcaaccagact 1380 gcctgtccag ccgactgcga ccccaacacc caggctagct gtgagtgccctgaaggctac 1440 atcctggacg acggtttcat ctgcacggac atcgacgagt gcgaaaacggcggcttctgc 1500 tccggggtgt gccacaacct ccccggtacc ttcgagtgca tctgcgggcccgactcggcc 1560 cttgcccgcc acattggcac cgactgtgac tccggcaagg tggacggtggcgacagcggc 1620 tctggcgagc ccccgcccag cccgacgccc ggctccacct tgactcctccggccgtgggg 1680 ctcgtgcatt cgggcttgct cataggcatc tccatcgcga gcctgtgcctggtggtggcg 1740 cttttggcgc tcctctgcca cctgcgcaag aagcagggcg ccgccagggccaagatggag 1800 tacaagtgcg cggccccttc caaggaggta gtgctgcagc acgtgcggaccgagcggacg 1860 ccgcagagac tctgagcggc ctccgtccag gagcctggct ccgtccaggagcctgtgcct 1920 cctcacccca gctttgctac caaagcacct tagctggcat tacagctggagaagaccctc 1980 cccgcacccc ccaagctgtt ttcttctatt ccatggctaa ctggcgagggggtgattaga 2040 gggaggagaa tgagcctcgg cctcttccgt gacgtcactg gaccactgggcaatgatggc 2100 aattttgtaa cgaagacaca gactgcgatt tgtcccaggt cctcactaccgggcgcagga 2160 gggtgagcgt tattggtcgg cagccttctg ggcagacctt gacctcgtgggctagggatg 2220 actaaaatat ttattttttt taagtattta ggtttttgtt tgtttcctttgttcttacct 2280 gtatgtctcc agtatccact ttgcacagct ctccggtctc tctctctctacaaactccca 2340 cttgtcatgt gacaggtaaa ctatcttggt gaattttttt ttcctagccctctcacattt 2400 atgaagcaag ccccacttat tccccattct tcctagtttt ctcctcccaggaactgggcc 2460 aactcacctg agtcacccta cctgtgcctg accctacttc ttttgctcttagctgtctgc 2520 tcagacagaa cccctacatg aaacagaaac aaaaacacta aaaataaaaatggccatttg 2580 ctttttcacc agatttgcta atttatcctg aaatttcaga ttcccagagcaaaataattt 2640 taaacaaagg ttgagatgta aaaggtatta aattgatgtt gctggactgtcatagaaatt 2700 acacccaaag aggtatttat ctttactttt aaacagtgag cctgaattttgttgctgttt 2760 tgatttgtac tgaaaaatgg taattgttgc taatcttctt atgcaatttccttttttgtt 2820 attattactt atttttgaca gtgttgaaaa tgttcagaag gttgctctagattgagagaa 2880 gagacaaaca cctcccagga gacagttcaa gaaagcttca aactgcatgattcatgccaa 2940 ttagcaattg actgtcactg ttccttgtca ctggtagacc aaaataaaaccagctctact 3000 ggtcttgtgg aattgggagc ttgggaatgg atcctggagg atgcccaattagggcctagc 3060 cttaatcagg tcctcagaga atttctacca tttcagagag gccttttggaatgtggcccc 3120 tgaacaagaa ttggaagctg ccctgcccat gggagctggt tagaaatgcagaatcctagg 3180 ctccacccca tccagttcat gagaatctat atttaacaag atctgcagggggtgtgtctg 3240 ctcagtaatt tgaggacaac cattccagac tgcttccaat tttctggaatacatgaaata 3300 tagatcagtt ataagtagca ggccaagtca ggcccttatt ttcaagaaactgaggaattt 3360 tctttgtgta gctttgctct ttggtagaaa aggctaggta cacagctctagacactgcca 3420 cacagggtct gcaaggtctt tggttcagct aagctaggaa tgaaatcctgcttcagtgta 3480 tggaaataaa tgtatcatag aaatgtaact tttgtaagac aaaggttttcctcctctatt 3540 ttgtaaactc aaaatatttg tacatagtta tttatttatt ggagataatctagaacacag 3600 gcaaaatcct tgcttatgac atcacttgta caaaataaac aaataacaatgtgaaaaaaa 3660 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 3693

What is claimed is:
 1. A method for treating a vascular disease in amammal, wherein said method comprising the steps of: infecting a segmentof a blood vessel in vitro using a gutless adenoviral vector comprisinga polynucleotide encoding a thrombomodulin protein or its variant;grafting the virus-treated blood vessel in said mammal, wherein saidthrombomodulin protein or its variant is expressed in said virus-treatedblood vessel in a amount sufficient to reduce re-occlusion or intimalhyperplasia in the grafted blood vessel.
 2. The method of claim 1,wherein said thrombomodulin protein has the amino acid sequence of SEQID NO:2.
 3. The method of claim 1, wherein said gutless adenoviralvector further comprises a regulatory element operably linked to the DNAsequence.
 4. The method of claim 3, wherein the regulatory element is aconstitutive promoter selected from a group consisting of CMV promoterand RSV promoter.
 5. The method of claim 1, wherein the expression ofsaid polynucleotide encoding a thrombomodulin protein or its variant isunder the control of an inducible system.
 6. The method of claim 1,wherein said gutless adenoviral vector is produced using a shuttlevector comprising a pBR322 replication origin, a selectable marker gene,an adenovirus left inverted terminal repeat, an adenovirus encapsidationsignal, an intronic sequence, and an adenovirus left inverted terminalrepeat.
 7. The method of claim 6, wherein said selectable marker gene isKanamycin resistance gene.
 8. The method of claim 1, wherein said mammalis human
 9. The method of claim 1, wherein said infecting step furthercomprises: filling the blood vessel with a complete viral deliverysystem comprising of 1:1 mixture of Ham's F12 medium and DMEM, aneffective amount of the gutless adenovirus vector, and an acellularoxygen carrier; and incubating the blood vessel with the complete viraldelivery system for a desired period of time.
 10. The method of claim 9,wherein said acellular oxygen carrier is selected from the groupconsisting of unmodified hemoglobin, chemically modified hemoglobin andperfluorochemical emulsions.
 11. The method of claim 10, wherein saidunmodified hemoglobin or chemically modified hemoglobin is used in therange of 3 g/dl to 10 g/dl.
 12. The method of claim 9, wherein thecomplete viral delivery system further comprises at least one ofL-glutamine, sodium bicarbonate, or antibiotic-antimycotic.
 13. Themethod of claim 9, wherein the desired period of time is between 10 to45 minutes.
 14. A method for treating a vascular disease in a mammal,wherein said method comprising the steps of: evacuating a clot from ablood vessel in said mammal; isolating a segment of the blood vesselaround the evacuation site; and infecting the segment of blood vessel invivo using a gutless adenoviral vector comprising a polynucleotideencoding a thrombomodulin protein or its variant; wherein thethrombomodulin protein or its variant is expressed in a amountsufficient to reduce re-occlusion or intimal hyperplasia in the infectedsegment of the blood vessel.
 15. The method of claim 14, wherein theisolating step further comprises the steps of: inserting a ballooncatheter to the site of evacuation; and inflating a proximal balloon anda distal balloons to isolate the vessel segment around the site ofevacuation.
 16. The method of claim 14, wherein said infecting stepfurther comprises the steps of: filling the isolated vessel segment witha complete viral delivery system comprising of 1:1 mixture of Ham's F12medium and DMEM, an effective amount of the gutless adenovirus vector,and an acellular oxygen carrier; and incubating the isolated vesselsegment with the complete viral delivery system for a desired period oftime.
 17. The method of claim 14, wherein said thrombomodulin proteinhas an amino acid sequence of SEQ ID NO:2.
 18. The method of claim 14,wherein said gutless adenoviral vector comprises a regulatory elementoperably linked to a DNA sequence encoding a thrombomodulin protein or avariant of the thrombomodulin protein.
 19. The method of claim 18,wherein said regulatory element is a constitutive promoter selected froma group consisting of CMV promoter and RSV promoter.
 20. The method ofclaim 14, wherein said polynucleotide encoding a thrombomodulin proteinor its variant is under the control of an inducible system.
 21. Themethod of claim 14, wherein said gutless adenoviral vector is producedusing a shuttle vector comprising a pBR322 replication origin, aselectable marker gene, an adenovirus left inverted terminal repeat, anadenovirus encapsidation signal, an intronic sequence, and an adenovirusleft inverted terminal repeat.
 22. The method of claim 14, wherein saidmammal is human.
 23. A method for treating a vascular disease in amammal comprising administering a therapeutically effective amount of agutless adenovirus vector into a segment of a blood vessel using astent, wherein said gutless adenovirus vector is capable of expressing athrombomodulin protein or a variant of the thrombomodulin protein. 24.The method of claim 23, wherein said thrombomodulin protein has an aminoacid sequence of SEQ ID NO:2.
 25. The method of claim 23, wherein saidgutless adenovirus vector is embedded in said stent and is released onlyat a treatment site.
 26. A composition for treating a vascular disease,comprising: a gutless adenovirus capable of expressing thrombomodulinprotein or a variant of the thrombomodulin protein, said gutlessadenovirus is produced using a shuttle vector comprising a pBR322replication origin, a selectable marker gene, an adenovirus leftinverted terminal repeat, an adenovirus encapsidation signal, anintronic sequence, and an adenovirus right inverted terminal repeat. 27.A pharmaceutical composition for treating a vascular disease accordingto claim 26, further comprising a pharmaceutically acceptable carrier.